Luciferase Assay for L1 Promoter and ASP

L1 promoter and ASP assays were conducted as described^{23 (link)} with some modifications. Briefly, 293T cells were cotransfected with pGLuc-5′-UTR, pCMV-CLuc (New England BioLabs), and pcDNA-Cas9m4-VP64, together with a plasmid expressing mock sgRNA or sgL1ASP using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). At 2 days after transfection, the Gaussia and Cypridina luciferase activities were measured using Gaussia or Cypridina Luciferase Assay Kits (New England BioLabs) according to the manufacturer’s instructions. The Gaussia luciferase (GLuc) activity was normalized to the corresponding Cypridina luciferase (CLuc) activity.

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Honda T., Nishikawa Y., Nishimura K., Teng D., Takemoto K, & Ueda K. (2020). Effects of activation of the LINE-1 antisense promoter on the growth of cultured cells. Scientific Reports, 10, 22136.

Publication 2020

pCMV-CLuc Lipofectamine 2000 reagent

293t cells Assay Cypridina luciferase Lipofectamine 2000 Luciferase Plasmid Transfection

Corresponding Organization : Osaka University

Other organizations : Kyoto University

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Variable analysis

independent variables

Plasmid expressing mock sgRNA or sgL1ASP

dependent variables

Gaussia luciferase (GLuc) activity
Cypridina luciferase (CLuc) activity

control variables

293T cells
PGLuc-5′-UTR plasmid
PCMV-CLuc plasmid
PcDNA-Cas9m4-VP64 plasmid
Lipofectamine 2000 reagent
Gaussia Luciferase Assay Kits
Cypridina Luciferase Assay Kits

controls

Positive control: pCMV-CLuc plasmid
Negative control: Mock sgRNA

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